Project Summary This application addresses the New Directions in Hematology Research (SHINE-II) Program ( PAS-15-168 ). The title of this proposal is ?Identifying the Function of the Mitochondrial Heme Biosynthesis Complex in Erythropoiesis.? This proposal addresses the molecular biology of heme during erythropoiesis as well as erythroid cell biology, iron metabolism and acquired and congenital disorders of red blood cell production. Heme is an essential protein cofactor required for erythrocyte development function. In all cells heme participates in many cellular reactions and also functions as a regulatory molecule for diverse cellular processes. While the reactions involved in heme synthesis have been well studied, little is known about the tissue specific regulation of heme synthesis and how heme homeostasis is maintained. Our objective is to determine how heme synthesis is regulated via protein complexes during erythropoiesis. We have recently identified a mitochondrial heme biosynthesis complex. Our preliminary data suggests that this complex is important for regulating heme synthesis and for protecting the cell from the reactive substrates and products of the pathway. Our hypothesis is that this mitochondrial heme biosynthesis complex is necessary for heme synthesis and specialized depending on the cell demand for heme. To understand the tissue specific regulation of heme synthesis by this complex, we will use cell culture models and zebrafish. We will utilize liver and adrenal cell culture models to identify the mitochondrial heme synthesis complex in these cells. We will compare this to our previous findings in an erythroid cell model for the identification of protein components unique for erythropoiesis. To investigate the role of novel components for the mitochondrial heme biosynthesis complex we will create cell lines and zebrafish in which select components of the complex have been disrupted. We will characterize erythroid cell development in these null lines and organisms to understand the role of these proteins in heme synthesis. Understanding how heme synthesis is regulated at the cellular level and defining unique components necessary for heme synthesis in developing erythroid cells will result in new directions of inquiry which will open up new pathways to discovery for pathological conditions including anemias, porphyrias and polycythemias.